Highly active antiretroviral therapy (HAART) has had a major impact on the AIDS epidemic in industrially advanced nations;however, no eradication of human immunodeficiency virus type 1 (HIV-1) appears to be currently possible, in part due to the viral reservoirs remaining in blood and infected tissues. Moreover, we have encountered a number of challenges in bringing about the optimal benefits of the currently available therapeutics of AIDS and HIV-1 infection to individuals receiving HAART. They include (i) drug-related toxicities;(ii) partial restoration of immunologic functions once individuals developed AIDS;(iii) development of various cancers as a consequence of survival prolongation;(iv) flaming-up of inflammation in individuals receiving HAART or immune re-construction syndrome (IRS);and (v) increased cost of antiviral therapy. Such limitations and flaws of HAART are exacerbated by the development of drug-resistant HIV-1 variants. Successful antiviral drugs, in theory, exert their virus-specific effects by interacting with viral receptors, virally encoded enzymes, viral structural components, viral genes, or their transcripts without disturbing cellular metabolism or function. However, at present, no antiretroviral drugs or agents are likely to be completely specific for HIV-1 or to be devoid of toxicity or side effects in the therapy of AIDS, which has been a critical issue because patients with AIDS and its related diseases will have to receive antiretroviral therapy for a long period of time, perhaps for the rest of their lives. Thus, the identification of new class of antiretroviral drugs which have an unique mechanism(s) of action and produce no or minimal side effects remains an important therapeutic objective. We recently designed, synthesized, and identified a novel non-peptidic PI, GRL-02031, by incorporating a stereochemically defined fused cyclopentanyltetrahydrofuran (Cp-THF), which exerted potent activity against a wide spectrum of HIV-1 including multi-drug-resistant HIV-1 variants (HIVMDR). GRL-02031 is highly potent against laboratory HIV-1 strains and primary clinical isolates including subtypes A, B, C, and E (50% effective concentration [EC50], 0.015-0.038 mM) with minimal cytotoxicity (CC50: &gt;100 mM in CD4+ MT-2 cells), although it was less active to two HIV-2 strains (HIV-2EHO and HIV-2ROD) (EC50: 0.60 mM) than to HIV-1 strains. GRL-02031 at relatively low concentrations blocks the infection and replication of each of HIV-1NL4-3 variants exposed to and selected by various commercially available PIs (EC50: 0.036 - 0.14 mM). GRL-02031 was also potent against multi-PI resistance in clinical HIV-1 variants isolated from patients who had no response to then existing conventional antiretroviral regimens with EC50 values ranging 0.014 - 0.042 mM (&lt;2-fold changes in EC50 values compared with that of wild-type HIV-1). Structural modeling analysis demonstrated a distinct bimodal binding of GRL-02031 to protease, which may provide advantages to GRL-02031 in blocking the replication of a wide spectrum of HIV-1 variants resistant to other currently available PIs and in delaying resistance development of HIV-1 to GRL-02031. These data warrant that GRL-02031 be further developed as a potential therapeutic agent for treatment of infection with primary and multi-drug-resistant HIV-1. We also generated a group of novel PIs, containing stereochemically defined flexible cyclic ethers/polyethers as the high affinity P2-ligands (1,3-dioxacycloalkanes), which exert potent enzyme inhibitory and antiviral activity. One such PI, (1S,2R)-1-Benzyl-2-hydroxy-3-[isobutyl(4-methoxybenzenesulfonyl)amino]propylcarbamic acid (1R)-3,5-dioxacycloheptan-1-yl ester, showed an enzymatic Ki of 26 pM and antiviral IC50 value of 4.6 nM in MT-2 human T-lymphoid cells exposed to HIV-1LAI.We previously identified a new class of nucleoside reverse transcriptase inhibitors (NRTIs), 4-ethynyl-2-deoxunucleoside analogs (EdNs), which, unlike conventional NRTIs such as azidothymidine, contain the 3-hydroxy moiety. These EdNs exert potent antiviral activity agains a wide spectrum of HIV-1 and HIV-2 strains. In the time period of the annual report, we examined the in vivo activity of 4-ethynyl-2-fluoro-2-deoxyadenosine (EFdA), one of the EdNs, which has a potent activity against various HIV-1 and simian immunodeficiency virus (SIV) strains including multi-drug resistant HIV-1 variants and is thought to be suitable for once daily regimen, in two SIV-infected monkeys, in collaboration with Dr. Michael Parniak. EFdA treatment resulted in a 2 -3 log decrease in SIV load within 7 days;these levels declined to undetectable (5-log reduction) within 2 months and essentially remained so for the duration of therapy. SAIDS symptoms (chronic diarrhea, dehydration) resolved within 1 month of EFdA treatment. The animals showed steady weight gain and retained normal liver enzyme levels throughout the course of EFdA treatment. Animal R395 was sacrificed after 3 months of EFdA therapy due to unrelated respiratory distress. SIV RNA was undetectable in most tissues in this animal, and only low levels (&lt;100 copies) were noted in a few CD4+ T-cell rich lymph nodes, suggesting that EFdA therapy was able to clear SIV from many tissue reservoirs despite only 3 months of drug treatment. Such apparent clearance was not noted in animal R393 as plasma SIV levels rebounded following the 6-month treatment. This pilot study shows that EFdA is safe and very effective in reducing plasma virus load and in resolving SAIDS symptoms. This study shows that QD administration of EFdA is very effective in reducing plasma virus load and in resolving SAIDS symptoms in SIV-infected macaques, and achieved substantial virus clearance in one animal. The drug appeared safe as no hepatoxicity was seen over the 6-month course of treatment. The data from this pilot study suggest that more extensive non-human primate evaluations of EFdA antiretroviral efficacy are warranted.